The postsynaptic density (PSD) at excitatory glutamatergic synapses is a large molecular machine that is known to be a key site of information processing and storage. In order to explore the detailed molecular organization of the PSD, we developed a method to freeze-substitute hippocampal cultures and then examine them in thin sections by EM tomography to show individual protein complexes in their natural setting within the PSD. The initial work employing tomography revealed that the core of the PSD is an array of vertically oriented filaments that contain the scaffold protein, PSD-95, in an extended configuration and a polarized orientation, with its N-terminus positioned at the postsynaptic membrane. This finding provided insight into the overall organization of the PSD because scaffolding proteins, such as PSD-95 family MAGUK proteins, have distinct multiple, diverse binding sites for other proteins arrayed along their length. Thus, the regular arrays of PSD-95 impose an ordering on many other PSD proteins, including the glutamate receptors, and provide an overall plan for the structure of the PSD. Mechanisms that regulate PSD-95 MAGUK conformations were investigated in collaboration with the William Green laboratory. Fluorescent Resonate Energy Transfer (FRET) was applied to measure PSD-95 molecules in different functional configurations. PSD-95 adopts an extended conformation in PSDs, but remains in closed conformation at non-synaptic sites. In contrast, SAP-97, another MAGUK, adopts an open configuration oriented parallel with the post synaptic membrane. The open conformation of PSD-95 at the PSD is now established as a requirement for it to interact with NMDAR and AMPAR-Stargazin complexes. EM tomography also revealed that the C-terminal ends of the PSD-95 vertical filaments are associated with horizontally oriented filaments. Immunogold labeling identifies one class of horizontal filaments as GKAP, which is known to bind to the GK domain at the C-terminal end of PSD-95. The emerging structural model of the PSD shows how the PSD-95 matrix can stabilize glutamate receptors forming elaborate molecular complexes, and at the same time, allows room for the addition of new receptors at the edges of the PSD. We have used antibodies to label proteins in tomograms, but the antibody complexes appearing as filamentous structures in tomograms confounds the identification of the target protein. An indirect approach combining knockdown with tomography has proved additional, but provided limited information for identifying components of protein complexes in tomograms. We attempted to develop an additional alternative method for identifying the proteins using an expressible probe, miniSOG, which generates singlet oxygen upon blue light illumination, and then oxidizes diaminobenzidine (DAB) to form electron dense polymers visualized by EM. We overexpressed miniSOG-PSD-95 in neurons and found that the staining generated by miniSOG is too diffuse to localize molecules with better than 20 nm precision. We therefore used various dilutions of sucrose solution in the photoconversion process to slowdown molecular diffusion. Eventually, with the help of EM tomography, we found evidence that electron dense structures developed at the distal ends of some of the membrane associated vertical filaments known to contain PSD-95, thus allowed identification of miniSOG tagged PSD-95. We plan to expand this work by making CaMKII-miniSOG construct to see if the similar results will provide a specific marker for the large macromolecule CaMKII. The idea that the PSD-95 dependent scaffold stabilizes the PSD has been explored by using EM tomography to determine the effects of RNAi knock down of MAGUKs. We examined the effects of knocking down, simultaneously, three major MAGUK proteins: PSD-95, PSD-93 and SAP102, and EM tomography revealed significant loss from the central core of the PSD, including NMDA receptor structures, vertical filaments, and AMPA receptors. Electrophysiology measurements by collaborators from the Roger Nicoll laboratory characterizing the effects of the same knock down show significant functional loss of NMDAR and AMAPR type EPSPs at levels compatible with the structural losses. Electron microscopy also showed depletion of vertical filaments along with AMPAR type structures at the peripheral region of the PSD, and significant reduction in size of NMDAR clusters in the center of the PSD. These structural data indicate that vertical filaments corresponding to MAGUKs anchor AMPARs and are also a factor in organizing NMDARs. Thus, PSD-95 MAGUKs are demonstrated to be the essential organizer of glutamate receptors at the PSD. We also developed another line of research recently with the Rumbaugh Lab to characterize structural role of SynGAP, which negatively regulates AMPAR binding to the PDZ domain of PAS-95 MAGUKs at the PSD. We are also trying to identify NMDARs more directly in intact hippocampal synapses by using CRISPR-Cas9 construct developed in the Nicoll Lab to knockout the obligated GluN1 subunit of NMDARs and reconstruction of the postsynaptic density (PSD) with dark field scanning EM tomography. We now have confirmation that the class of transmembrane structures containing larger globular cytoplasmic profile likely contain NMDARs, whereas the structures with smaller and flat cytoplasmic profile likely contain AMPARs. We are studying with M. DellAcqua, University of Colorado, the conformations and distribution of A Kinase Anchoring Proteins (AKAPs) hippocampal synapses. These molecules are membrane associated proteins known to interact with PSD-95 MAGUKs and anchor several classes of kinases (PKA, PKC) and calcineurin, important for synaptic plasticity (LTP and LTD). This work is showing that there is a conformational change in AKAPs in the PSD, different from that at the extrasynaptic membrane. This distinction may have important functional implications in understanding the role of AKAPs in regulating AMPARs at the PSDs. In collaboration with the Roger Nicoll Lab, we are studying the effects of over expressing constitutively activated CaMKII on synaptic structure and function. Electrophysiology measurements show that activated CaMKII expression enhances synaptic transmission, and we plan to analyze changes in spine sizes and PSD structure, using serial section EM or thick section STEM tomography. Finally, we have developed a new electron microscopic method in collaboration with Richard Leapman using darkfield STEM tomography for sections up to 300-400 nm thick to provide detailed reconstructions of whole PSDs. The darkfield imaging, which provides enhanced visualization of the smallest structures at the PSDs, provides an opportunity to reconstruct detailed molecular organization of more or less complete PSDs in intact neurons. A new initiative is an ongoing collaboration with Carolyn Smith in the NINDS Light Microscopy Facility. Dr. Smith has cultured a primitive animal, Trichoplax, that is remarkable in that it lacks synapses, but shows behavior indicative of neural function. These results appear to signify an early stage in evolving nervous systems, prior to the development of synapses, that utilizes peptide signaling pathways dependent on many of the same proteins found at synapses in higher animals. A cell that senses direction of gravity and mediates behavior accordingly has also been discovered, but what control systems are utilized is not yet clear. Knowing exactly how these unconventional, nonsynaptic systems function to control behaviors is expected to provide previously overlooked information on non-synaptic signaling mechanisms in mammalian brains. In the next year we plan to complete the several synaptic projects listed above.